猪繁殖与呼吸综合征病毒GP5a蛋白在大肠埃希菌中的表达  

Prokaryotic expression of porcine reproductive and respiratory syndrome virus GP5a protein gene in Escherichia coli

作　　者：徐彦召[1] 王青[1] 王秋霞[1] 杭柏林[1] 孙亚伟[1] 胡建和[1] 

机构地区：[1]河南科技学院,河南新乡453003

出　　处：《中国兽医学报》2016年第3期384-388,共5页Chinese Journal of Veterinary Science

基　　金：国家自然基金青年科学基金资助项目(31402208);河南省教育厅科学技术研究重点项目指导计划资助项目(14B230003)

摘　　要：构建高致病性猪繁殖与呼吸综合征病毒(highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)HuN4株GP5a蛋白基因的原核表达载体,并将其在宿主菌BL21(DE3)感受细胞中进行表达。以HP-PRRSV HuN4株GP5a蛋白的基因序列为模板,通过RT-PCR扩增获得GP5a蛋白全长基因片段,将其定向克隆到原核表达载体pGEX-6P-1中,构建含有GP5a蛋白基因的重组表达载体pGEX-GP5a;将构建的重组质粒转化宿主菌,经IPTG诱导,表达出目的蛋白;表达蛋白采用Western blot方法检测其反应原性。结果显示,成功克隆出了HP-PRRSV HuN4株GP5a蛋白基因全长,片段序列为156bp;构建的pGEX-GP5a载体经PCR、双酶切、测序鉴定均正确,转化表达宿主菌后经SDS-PAGE检测目的蛋白以包涵体的形式表达,用包涵体纯化法获得该蛋白;Western blot检测表达蛋白的反应原性,结果显示表达蛋白具有良好的反应原性。本试验成功构建了含有HP-PRRSV HuN4株GP5a蛋白基因的原核表达载体,成功表达、纯化得到了GP5a蛋白,为进一步研究GP5a蛋白的功能提供材料。The GP5 aprotein gene of highly pathogenic porcine reproductive and respiratory syndrome virus（HP-PRRSV）was cloned into a prokaryotic expression vector（pGEX-6P-1）,and then using Escherichia coli（E.coli）BL21（DE3）to express GP5 aprotein.GP5aprotein gene was amplified from total RNA which was reverse transcription of HP-PRRSV HuN4 strain,then the gene was cloned into pGEX-6P-1;PCR,digestion and sequencing were used to identify positive plasmid,which named pGEX-GP5a;the identified recombined plasmid was expressed by E.coli BL21（DE3）and induced by IPTG.SDS-PAGE was used to analyze the expressed protein,and the Western blot was used to identify the reaction activity.The results showed that the sequence of GP5 aprotein gene was consistent with predicted size,156 bp.PCR,digestion and sequencing results showed that the recombinant plasmid pGEX-GP5 awas correct.GP5 aprotein was successfully expressed by E.coli BL21（DE3）,while GP5 aprotein was expressed in DE3 cells as an inclusion body,and the recombinant protein was harvested by the inclusion body purification techniques.The recombinant protein could react to the antibody of HP-PRRSV HuN4 strain using the method of Western blot assay.In summary,the recombinant plasmid pGEX-GP5 acontaining GP5aprotein gene was constructed,and recombinant protein was successful expressed in E.coli BL21（DE3）.

关 键 词：高致病性猪繁殖与呼吸综合征病毒 GP5a蛋白 原核表达 

分 类 号：S852.651[农业科学—基础兽医学]